Improvement of the optimum temperature of lipase activity for Rhizopus niveus by random mutagenesis and its structural interpretation

Random mutagenesis was used to improve the optimum temperature for Rhizopus niveus lipase (RNL) activity. The lipase gene was mutated using the error- prone PCR technique. One desirable mutant was isolated, and three amino aci ds were substituted in this mutant (P18H, A36T and E218V). The wild-type an d this randomly mutated lipase were both purified and characterized. The sp ecific activity of the mutant lipase was 80% that of the wild-type. The opt imum temperature of the mutant lipase was higher by 15 degreesC than that o f the wild-type. To confirm which substitution contributed to enhancing the optimum temperature for enzymic activity, two chimeric lipases from the wi ld-type and randomly mutated gene were constructed: chimeric lipase 1 (CL-1 ; P18H and A36T) and chimeric lipase 2 (CL-2; E218V). Each of the chimeric enzymes was purified, and the optimum temperature for lipase activity was m easured. CL-1 had a similar optimum temperature to that of the wild-type, a nd CL-2 had a higher temperature like the randomly mutated lipase. The muta tional effect is interpreted in terms of a three-dimensional structure for the wild-type lipase, (C) 2001 Elsevier Science B.V. All rights reserved.